Primers for detecting serotypes of Shigella flexneri and multiplex amplifications using the same

ABSTRACT

The present inventions relates to primers for identifying  Shigella flexneri  serotypes comprising the sequences of SEQ ID Nos. 1 and 2, SEQ ID Nos. 3 and 4, SEQ ID Nos. 5 and 6, SEQ ID Nos. 7 and 8, SEQ ID Nos. 9 and 10, SEQ ID Nos. 11 and 12, SEQ ID Nos. 13 and 14, SEQ ID Nos. 15 and 16. These primers are specific and have a common annealing temperature. The present invention further relates to a multiplex amplification-based identification method using the primers. The present invention further relates to the use of the primers for identifying  Shigella flexneri  serotypes for the preparation of identification agents. The present invention further relates to a kit for identifying  Shigella flexneri  comprising the above primers.

REFERENCE TO SEQUENCE LISTING SUBMITTED ELECTRONICALLY

The content of the electronically submitted sequence listing, file name:3503_0010001_Sequence_Listing.txt; size: 13,842 bytes; and date ofcreation: Oct. 22, 2014, filed herewith, is incorporated herein byreference in its entirety.

TECHNICAL FIELD

The present invention relates to the field of biotechnology, morespecifically, to primers for identifying serotypes of Shigella flexneriand to multiplex amplifications using the same.

BACKGROUND OF THE INVENTION

Shigella species are the major pathogenic bacteria causing bacterialdiarrhea in developing countries, which infect 164.7 million people andlead to 0.11 million deaths per year, most of which are children under 5years old (Kotloff, K. L., J. P. Winickoff, B. Ivanoff, J. D. Clemens,D. L. Swerdlow, P. J. Sansonetti, G. K. Adak, and M. M. Levine. 1999.Global burden of Shigella infections: implications for vaccinedevelopment and implementation of control strategies. Bull World HealthOrgan 77:651-66). Among the four serogroups of Shigella, Shigellaflexneri is the predominant serogroup that affects the low-incomepopulation.

Shigella flexneri is further classified into various serotypes accordingto the different 0-antigen structures. To date, at least 15 serotypeshave been reported, i.e. 1a, 1b, 1c, 2a, 2b, 3a, 3b, 4a, 4b, 5a, 5b, X,Xv, Y, and F6 (Simmons, D. A., and E. Romanowska. 1987. Structure andbiology of Shigella flexneri O antigens. J. Med. Microbiol. 23:289-302;Stagg, R. M., S. S. Tang, N. I. Carlin, K. A. Talukder, P. D. Cam, andN. K. Verma. 2009. A novel glucosyltransferase involved in O-antigenmodification of Shigella flexneri serotype 1c. J. Bacteriol. 191:6612-7;Ye, C., R. Lan, S. Xia, J. Zhang, Q. Sun, S. Zhang, H. Jing, L. Wang, Z.Li, Z. Zhou, A. Zhao, Z. Cui, J. Cao, D. Jin, L. Huang, Y. Wang, X. Luo,X. Bai, P. Wang, Q. Xu, and J. Xu. 2010. Emergence of a newmultidrug-resistant serotype X variant in an epidemic clone of Shigellaflexneri. J. Clin. Microbiol. 48:419-26). Serotyping has long been useto characterize isolates for epidemiological purposes

The LPSs of all Shigella flexneri serotypes except F6 have the samepolysaccharide backbone consisting of repeating tetrasaccharide units,and serotype Y has the basic tetrasaccharide backbone (Simmons, D. A.,and E. Romanowska. 1987. Structure and biology of Shigella flexneri Oantigens. J. Med. Microbiol. 23:289-302). Modification by glycosylationand/or acetylation of different sugars on the backbone gives rise tovarious type-specific antigenic determinants (e.g., I, II, III, IV, V,and VI), group-specific antigenic determinants (e.g., 3,4; 6; and 7,8),and antigenic determinant 1c (Stagg, R. M., S. S. Tang, N. I. Carlin, K.A. Talukder, P. D. Cam, and N. K. Verma. 2009. A novelglucosyltransferase involved in O-antigen modification of Shigellaflexneri serotype 1c. J. Bacteriol. 191:6612-7).

Three genes (gtrA, gtrB, and gtr_([type])) are responsible forglycosylation modifications on Shigella flexneri. The first two genesare highly homologous and interchangeable, whereas the third genegtr_([type]) is unique, and encodes serotype-specificglycosyltransferases (Allison, G. E., and N. K. Verma. 2000.Serotype-converting bacteriophages and O-antigen modification inShigella flexneri. Trends. Microbiol. 8:17-23; Stagg, R. M., S. S. Tang,N. I. Carlin, K. A. Talukder, P. D. Cam, and N. K. Verma. 2009. A novelglucosyltransferase involved in O-antigen modification of Shigellaflexneri serotype 1c. J. Bacteriol. 191:6612-7). Gtr genes specific totype-antigens I, II, IV and V, group antigen 7,8 and antigen 1c aregtrI, gtrII, gtrIV, gtrV, gtrX and gtrIC, respectively (Adams, M. M., G.E. Allison, and N. K. Verma. 2001. Type IV O antigen modification genesin the genome of Shigella flexneri NCTC 8296. Microbiology 147:851-60;Adhikari, P., G. Allison, B. Whittle, and N. K. Verma. 1999. Serotype 1aO-antigen modification: molecular characterization of the genes involvedand their novel organization in the Shigella flexneri chromosome. JBacteriol 181:4711-8; Guan, S., D. A. Bastin, and N. K. Verma. 1999.Functional analysis of the O antigen glucosylation gene cluster ofShigella flexneri bacteriophage SfX. Microbiology 145: 1263-73; Huan, P.T., D. A. Bastin, B. L. Whittle, A. A. Lindberg, and N. K. Verma. 1997.Molecular characterization of the genes involved in O-antigenmodification, attachment, integration and excision in Shigella flexneribacteriophage SfV. Gene 195:217-27; Mavris, M., P. A. Manning, and R.Morona. 1997. Mechanism of bacteriophage SfII-mediated serotypeconversion in Shigella flexneri. Mol Microbiol 26:939-50; Stagg, R. M.,S. S. Tang, N. I. Carlin, K. A. Talukder, P. D. Cam, and N. K. Verma.2009. A novel glucosyltransferase involved in O-antigen modification ofShigella flexneri serotype 1c. J Bacteriol 191:6612-7). The gtr genesare carried by prophages integrated in the genome of host bacteria.O-acetylation, which confers group-antigen 6 and/or type-antigen III onstrains of serotypes 1b, 3a, 3b and 4b, is mediated by the oac genecarried in bacteriophage Sf6 (Clark, C. A., J. Beltrame, and P. A.Manning. 1991. The oac gene encoding a lipopolysaccharide O-antigenacetylase maps adjacent to the integrase-encoding gene on the genome ofShigella flexneri bacteriophage Sf6. Gene 107:43-52; Verma, N. K., J. M.Brandt, D. J. Verma, and A. A. Lindberg. 1991. Molecularcharacterization of the O-acetyl transferase gene of convertingbacteriophage Sf6 that adds group antigen 6 to Shigella flexneri. Mol.Microbiol. 5:71-5). Strains of different serotypes carry one or moreserotype-specific prophages that encode different specific 0-antigenmodifications genes (see FIG. 1).

Currently, the method for routinely identifying Shigella flexneriserotypes is the slide agglutination method using rabbit-originatedantiserum raised against Shigella flexneri-specific type- andgroup-factors. Commercially available diagnostic sera have been widelyused in microbiology laboratories. However, such a method has somedisadvantages. Firstly, to identify the serotype of one Shigella strain,the slide agglutination method requires as many as 10 reaction tests,using antibodies against type-antigens I, II, III, IV, V and VI,antibodies against group-antigens 3,4 and 7,8, and the monoclonalantibody against serotype 1c (MASF1c), respectively (Stagg, R. M., P. D.Cam, and N. K. Verma. 2008. Identification of newly recognized serotype1c as the most prevalent Shigella flexneri serotype in northern ruralVietnam. Epidemiol Infect 136:1134-40; Talukder, K. A., Z. Islam, M. A.Islam, D. K. Dutta, A. Safa, M. Ansaruzzaman, A. S. Faruque, S. N.Shahed, G. B. Nair, and D. A. Sack. 2003. Phenotypic and genotypiccharacterization of provisional serotype Shigella flexneri 1c and clonalrelationships with 1a and 1b strains isolated in Bangladesh. J ClinMicrobiol 41:110-7; Ye, C., R. Lan, S. Xia, J. Zhang, Q. Sun, S. Zhang,H. Jing, L. Wang, Z. Li, Z. Zhou, A. Zhao, Z. Cui, J. Cao, D. Jin, L.Huang, Y. Wang, X. Luo, X. Bai, P. Wang, Q. Xu, and J. Xu. 2010.Emergence of a new multidrug-resistant serotype X variant in an epidemicclone of Shigella flexneri. J Clin Microbiol 48:419-26). Secondly,visual assessment of the slide agglutination reactions may deliverincorrect readings. Thirdly, expensive antiserum kits limit theapplication of such a method in laboratories in developing countries.

Therefore, development of a rapid, specific identification method basedon biomolecular technologies such as PCR will be significant for timelyand accurate identification of Shigella flexneri serotypes.

SUMMARY OF THE INVENTION

One objective of the present invention is to provide a set of primersfor identifying Shigella flexneri serotypes so as to identify Shigellaflexneri serotypes by multiplex amplification, in acknowledgement of theO-antigen modification genes of Shigella flexneri.

Another objective of the present invention is to provide the use of theprimers, in particular, for identification of Shigella flexneriserotypes and for preparation of agents for identifying Shigellaflexneri serotypes.

Yet another objective of the present invention is to provide a methodfor identifying Shigella flexneri serotypes by multiplex amplificationusing the primers.

Still another objective of the present invention is to provide agentsfor identifying Shigella flexneri serotypes, such as a kit.

In one aspect of the present' invention, a set of primers for multiplexamplification used for identification of Shigella flexneri serotypes isdesigned based on genes gtrI, gtrII, oac, gtrIV, gtrV, gtrX, wzx₁₋₅ andgtrIC. The set of primers includes amplification primers for identifyingtarget genes gtrI, gtrII, oac, gtrIV, gtrV, gtrX, wzx₁₋₅ and gtrIC. In aparticular embodiment of the present invention, amplification primershaving specificity and a common annealing temperature are designed basedon known sequences of genes gtrI, gtrII, oac, gtrIV, gtrV, gtrX, wzx₁₋₅and gtrIC, and more specifically, are the following amplificationprimers of the present invention: SEQ ID Nos. 1 and 2, for amplificationof the gtrI gene fragment; SEQ ID Nos. 3 and 4, for amplification of thegtrII gene fragment; SEQ ID Nos. 5 and 6, for amplification of the oacgene fragment; SEQ ID Nos. 7 and 8, for amplification of the gtrIV genefragment; SEQ ID Nos. 9 and 10, for amplification of the gtrV genefragment; SEQ ID Nos. 11 and 12, for amplification of the gtrX genefragment; SEQ ID Nos. 13 and 14, for amplification of the wzx₁₋₅ genefragment; and SEQ ID Nos. 15 and 16, for amplification of the gtrIC genefragment.

The primers of the invention can be used for qualitative identificationof Shigella flexneri serotypes. All currently known serotypes can besuccessfully identified, except for serotype Xv, which cannot bedistinguished from serotype X.

According to a preferred embodiment of the present invention, the set ofprimers for identifying Shigella flexneri serotypes provided by thepresent invention may further include a primer pair specific to F6. Theaverage isolation rate of serotype F6 in Asia is 6%, ranging from 0%(China) to 15% (Pakistan) (von Seidlein, L., D. R. Kim, M. Ali, H. Lee,X. Wang, V. D. Thiem, G. Canh do, W. Chaicumpa, M. D. Agtini, A.Hossain, Z. A. Bhutta, C. Mason, O. Sethabutr, K. Talukder, G. B. Nair,J. L. Deen, K. Kotloff, and J. Clemens. 2006. A multicentre study ofShigella diarrhoea in six Asian countries: disease burden, clinicalmanifestations, and microbiology. PLoS Med 3:e353). It is necessary tointroduce an F6-specific singleplex PCR for countries where F6 isprevalent. However, since the multiplex amplification primers of thepresent invention already contain 8 pairs of primers, adding anadditional pair of primers increases the difficulty in optimizing themultiplex PCR. Thus, one has to be cautious when interpreting theamplification-negative strains in the multiplex PCR as serotype F6.According to a preferred embodiment of the present invention, asingleplex PCR is employed to confirm F6, and the primer pair foridentifying F6 is SEQ ID Nos. 17 and 18 for amplification of the wzx₆gene fragment. Furthermore, as the strains have been verified withpolyvalent antisera for Shigella and serogroup B prior to the multiplexPCR, the probability of false-positive F6 is low.

In another aspect, the present invention further provides the use of theprimers, particularly including the use of the primers foridentification of Shigella flexneri serotypes and the use of the primersfor preparation of agents for identifying Shigella flexneri serotypes.

Based on the use of the primers for identifying Shigella flexneriserotypes provided by the present invention, the present invention alsoestablishes a set of rapid and sensitive methods ready forhigh-throughput application, for identifying Shigella flexneri serotypesby amplification. In particular, the method is a multiplex amplificationidentification method, comprising carrying out identification bymultiplex amplification using the primers of the present invention,wherein the amplification is preferably polymerase chain reaction (PCR).Since the multiplex amplification technique involves multiple pairs ofprimers and multiple pairs of templates as compared with conventionalamplification techniques, it is influenced by more factors and thus hasa higher chance to give mismatched amplification products. Therefore,the design of the primers is crucial. In the present invention, on thebasis of the designed multiple pairs of primers, the reaction system andconditions, especially the annealing temperature, are preferablyoptimized. In PCR, the PCR system is composed of heat-resistant DNApolymerases, primers, deoxynucleotides, DNA templates to be amplified,and a buffer. The present invention provides a preferred PCR reactionwhich is performed under the following conditions: pre-denaturation at95° C. for 15 min; 30 cycles of: denaturation at 94° C. for 30 s,annealing at 55° C. for 90 s, and extension at 72° C. for 60 s; andfinal extension at 72° C. for 10 min.

The multiplex amplification-based identification method of the presentinvention preferably further includes performing a qualitative analysisafter the amplification. The method for the qualitative analysis may bethose known to a person skilled in the art, e.g., a method includingvisualization of amplification products using gel electrophoresis, inwhich case a person skilled in the art can determine the gel and gelconcentrations to be used, according to the sizes of the amplificationproducts.

In order to validate the effectiveness of the method of the presentinvention, in particular embodiments of the present invention, theprimers of the present invention were used to conduct amplification onstrains representing 14 currently common Shigella flexneri serotypes bymultiplex PCR, and 358 Shigella flexneri strains of various serotypeswere analyzed. The multiplex PCR results for the assayed strains have aconcordance of 97.8% with those obtained by the slide agglutinationmethod. In order to assess the specificity of the primers of the presentinvention, 50 non flexneri strains, including other Shigella serogroupsand enteric pathogens, were also examined in particular embodiments ofthe present invention. These bacteria were all negative in theamplification, proving that the specificity of the method of the presentinvention is 100%. These results demonstrate that timely and accurateidentification of Shigella flexneri serotypes can be achieved by usingthe primers and the amplification-based identification method of thepresent invention.

According to the use of the primers in the preparation of agents foridentifying Shigella serotypes as provided in the present invention, theidentification agents of the present invention are preferably used toassay isolated Shigella flexneri strains. According to particularembodiments of the present invention, the identification agents may be akit. The identification agents may be used to assay Shigella flexneri orassay Shigella flexneri-containing samples used for identification anddiagnosis. In the present invention, the identification agents arepreferably used to assay excrement, intestinal effusions, and vomit. Themultiplex amplification primers and the multiplex amplification methodof the present invention are highly specific, can be used alone as arapid identification method to assay enriched or unenrinched samples,such as ex vivo samples from patients (for example, excrement,effusions, vomit, etc.) and water, soil, food, and cosmetics that maycarry Shigella flexneri, and can achieve rapid diagnosis andidentification of Shigella flexneri serotypes.

The present invention further provides a kit for identifying Shigellaflexneri serotypes, which contains the primers of the present invention.The kit may further comprise target-detecting probes for detectingtarget genes, and the probes are for example SEQ ID No.19, SEQ ID No.20,SEQ ID No.21, SEQ ID No.22, SEQ ID No.23, SEQ ID No.24, SEQ ID No.25 andSEQ ID No.26. The kit of the present invention can be used toqualitatively identify Shigella flexneri serotypes.

Among Shigella flexneri, serotypes X and Xv have the same amplificationpattern (wzx₁₋₅ and gtrX). Thus, they are classified as the sameserotype Xv or X by the multiplex PCR method. Serotype Xv has been oneof the most prevalent serotypes in China since 2002 (Ye, C., R. Lan, S.Xia, J. Zhang, Q. Sun, S. Zhang, H. Jing, L. Wang, Z. Li, Z. Zhou, A.Zhao, Z. Cui, J. Cao, D. Jin, L. Huang, Y. Wang, X. Luo, X. Bai, P.Wang, Q. Xu, and J. Xu. 2010. Emergence of a new multidrug-resistantserotype X variant in an epidemic clone of Shigella flexneri. J ClinMicrobiol 48:419-26), which was initially designated as 4c as it canreact with both anti-type IV sera and anti-group 7,8 sera (Pryamukhina,N. S., and N. A. Khomenko. 1988. Suggestion to supplement Shigellaflexneri classification scheme with the subserovar Shigella flexneri 4c:phenotypic characteristics of strains. J Clin Microbiol 26:1147-9).However, since factors responsible for type IV antigenic determinanthave not been clarified (Ye, C., R. Lan, S. Xia, J. Zhang, Q. Sun, S.Zhang, H. Jing, L. Wang, Z. Li, Z. Zhou, A. Zhao, Z. Cui, J. Cao, D.Jin, L. Huang, Y. Wang, X. Luo, X. Bai, P. Wang, Q. Xu, and J. Xu. 2010.Emergence of a new multidrug-resistant serotype X variant in an epidemicclone of Shigella flexneri. J Clin Microbiol 48:419-26), it is currentlyimpossible to develop a PCR method to identify serotype Xv. In order todifferentiate between serotypes Xv and X, an additional slideagglutination reaction using anti-type IV serum would be necessary.

Above all, according to the present invention, specific primers with acommon annealing temperature used for identifying Shigella flexneriserotypes have been designed, and a multiplex PCR identification methodbased on the genes of Shigella flexneri O antigen-modification enzymeshas been established. The multiplex PCR method of the present inventionmay be employed for biomolecular identification of Shigella flexneriserotypes, and the majority of currently known serotypes (14 out of 15)can be easily and specifically identified in only one reaction. Incomparison with the conventional slide agglutination method (requiringas many as 10 independent reactions), the multiplex PCR method of thepresent invention is time-saving, does not require expensive antisera,and is especially suitable for high-throughput identification. With thismethod, identification of 96 samples can be completed within 3.5 hoursand costs only 25% of that of the slide method. The amplificationprimers and the multiplex amplification method of the present inventionmay be used to complement the serum agglutination method foridentification of Shigella flexneri serotypes, and are practicallysignificant and highly valuable for timely and accurate identificationof serotypes of clinical isolates of pathogenic bacteria and forprevention and treatment of shigellosis.

DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the chemical composition and genes responsible for specificmodifications of the O antigens of various Shigella flexneri serotypes.

FIG. 2 shows the multiplex PCR amplification results of referencestrains of 14 serotypes. The results were obtained by using the methodof the present invention, wherein M1 and M2 are 150 bp and 100 bp DNALadder Markers (purchased from TaKaRa, Japan), respectively.

FIG. 3 shows the specificity assay results of 50 non-Shigella flexneristrains. In the results, M: marker; C: positive control; 1-2: Shigellasonnei, phases I, II; 3-14: Shigella dysenteriae serotypes 1-12; 15-32:Shigella boydii serotypes 1-18; 33-34: Enteroaggregative E. coli; 35-37:Enterohemorrhagic E. coli 0157:H7; 38: Enteroinvasive E. coli; 39:Enteropathogenic E. coli; 40: Enterotoxigenic E. coli; 41: UropathogenicE. coli; 42-43: E. coli K12; 44: L monocytogenes; 45: V. cholera; 46:Salmonella paratyphi A; 47-48: Salmonella paratyphi B; 49: Yersiniaenterocolitica; 50: Salmonella choleraesuis.

DETAILED DESCRIPTION

The present invention will be further described with reference to thefollowing examples and the figures, for better understanding of theinvention. The examples are only illustrative and are not in any waylimiting the scope of the present invention. In the examples, anyexperimental methods without detailed conditions specified areconventional methods with conventional conditions well known in the artor conditions recommended by the manufacturer.

EXAMPLES Materials and Methods

Strains: 14 known Shigella flexneri serotypes (except serotype 5b, seeTable 1 for details) were used in the examples to establish theconditions for performing the multiplex PCR method. 358 Shigellaflexneri strains in total (Table 2) were used to evaluate theeffectiveness of the multiplex PCR method of the present invention. Inorder to examine the cross-reactions of the primers in this study, 50strains from different genera were examined (S. Sonnei (n=2), S.dysenteriae (n=12, including all 12 serotypes), S. boydii (n=18,including all 18 serotypes), Enteroaggregative E. coli (n=2),Enterohemorrhagic E. coli O 157:H7 (n=3), Enteroinvasive E. coli (n=1),Enteropathogenic E. coli (n=1), Enterotoxigenic E. coli (n=1),Uropathogenic E. coli (n=1), E. coli K12 (n=2), L. monocytogenes (n=1),V. cholera (n=1), Salmonella paratyphi A (n=1), Salmonella paratyphi B(n=2), Yersinia enterocolitica (n=1), and Salmonella choleraesuis(n=1)). The serotypes of all the Shigella flexneri strains wereconfirmed with polyvalent antisera (purchased from Denka Seiken, Japan)and monoclonal antibodies (purchased from Reagensia AB, Sweden). AllChinese strains used in the examples were isolated from diarrheapatients in Chinese and were preserved in the Microbiology Laboratory,National Institute for Communicable Disease Control and Prevention,Chinese Center for Disease Control and Prevention (China CDC). Otherstrains were purchased from National Collection of Type Cultures (NCTC),UK.

Preparation of DNA Templates

DNA templates were directly obtained from bacteria clones by the boilingmethod. A single colony from an overnight culture on an LB plate wasfirstly put into 30 μl distilled water, boiled at 100° C. for 10 min,placed into an ice bath for 5 min, and centrifuged at 13,000×g, 4° C.for 10 min. The supernatant was used as the templates for PCRamplification.

PCR Primers

The PCR primers used in the examples are listed in Table 3, the primersfor amplification of the wzx gene were designed with reference to YayueLi et al. (Li, Y., B. Cao, B. Liu, D. Liu, Q. Gao, X. Peng, J. Wu, D. A.Bastin, L. Feng, and L. Wang. 2009. Molecular detection of all 34distinct O-antigen forms of Shigella. J Med Microbiol 58:69-81), andother primers were designed according to the sequences of Shigellaflexneri serotype-specific genes gtrI, gtrIC, gtrII, oac, gtrIV, gtrV,and gtrX.

All primers were synthesized by Sangon Biotech (Shanghai) ascommissioned, and dissolved in TL buffer (10 mM Tris-Cl, 1 mM EDTA, pH8.0) to have a final concentration of 50 μM.

PCR Amplification and Identification

Multiplex PCRs were carried out using a QIAGEN Multiplex PCR Kit(QIAGEN). Each PCR reaction mixture comprises 1×PCR Master Mix(containing HotStarTaq DNA polymerase, Multiplex PCR buffer, and dNTPMix), primers (0.2 μM each) and 3 μl DNA templates, and is 50 μl intotal. In the examples, PCR amplification was performed based on the PCRconditions provided by the kit instructions in which some multiplex PCRcycling parameters were further optimized, Le, pre-denaturation at 95°C. for 15 min; totally 30 cycles of denaturation at 94° C. for 30s,annealing at 55° C. for 90 s, extension at 72° C. for 60 s; and finalextension at 72° C. for 10 min. The amplification was performed in athermocycler from SENSO (Germany). 5 microliter of amplificationproducts was mixed with loading buffer, electrophoresed on a 1.5%agarose gel, and visualized using EB staining for results. Wherenecessary, the PCR products were directly sequenced or cloned into apMD20-T TA cloning vector (TaKaRa, Japan) for cloning and sequencing.

Experimental Results

In the examples, reference strains were firstly subjected to singleplexPCR with the primers, and as a results, every pair of primers can givethe expected fragments after amplification, namely 783 bp (wzx₁₋₅), 1122bp (gtrI), 518 bp (gtrIC), 1268 bp (gtrII), 604 bp (oac), 378 bp(gtrIV), 905 bp (gtrV) and 425 bp (gtrX), respectively. Sequencing ofthe PCR amplification products proved that the amplified fragments werecorrect. Afterwards, multiplex PCR was carried out according to theprotocols described above in Materials and Methods, in which we trieddifferent annealing temperatures from 54° C. to 63° C. and found thatthe highest yield was obtained at 55° C. The results showed thatdifferent serotypes have different amplification patterns (see FIG. 2and Table 1). The expected specific PCR products were obtained from theamplification of each serotype. The amplified fragments of differentsizes can be well resolved on a 1.5% gel, and with two different DNAmarkers, each PCR product can be correctly identified by size. Theamplification patterns of the reference strains are as follows: 1a(wzx₁₋₅, gtrI), 1b (wzx₁₋₅, gtrI, oac), 1c (wzx₁₋₅, gtrI, gtrIC), 2a(wzx₁₋₅, gtrII), 2b (wzx₁₋₅, gtrII, gtrX), 3a (wzx₁₋₅, oac, gtrX), 3b(wzx₁₋₅, oac), 4a (wzx₁₋₅, gtrIV), 4b (wzx₁₋₅, gtrIV, oac), 5a (wzx₁₋₅,gtrV), X/Xv (wzx₁₋₅, gtrX) and Y (wzx₁₋₅). wzx₁₋₅ may also be used as areference gene for PCR reactions since it is present in all Shigellaflexneri serotypes except serotype F6, F6 is negative in theamplification as its O-antigen synthesizing genes are completelydifferent from those of other serotypes, and does not have any genes formodifications (Cheah, K. C., D. W. Beger, and P. A. Manning. 1991.Molecular cloning and genetic analysis of the rfb region from Shigellaflexneri type 6 in Escherichia coli K-12. FEMS Microbiol Lett 67:213-8;Simmons, D. A., and E. Romanowska., 1987. Structure and biology ofShigella flexneri O antigens. J Med Microbiol 23:289-302).

In the examples, we attempted to incorporate a pair of specific primersfor F6 in the reaction. However, the additional pair of primersincreases the difficulty in optimization of the multiplex PCR since thereaction already contains 8 pairs of primers. Hence, no good resultswere obtained after experimentation with 3 pairs of such primers(designed according to the sequence of F6-specific O-antigen gene wzx₆),and one should be cautious when interpreting a strain negative in theamplification as serotype F6. According to preferred embodiments of thepresent invention, a singleplex PCR was used to confirm F6, the primersfor which can be seen in Table 3, and the length of the amplifiedproduct is 739 bp. Furthermore, since all strains have been verifiedwith polyvalent antisera for Shigella and serogroup B, possibility of afalse-positive F6 is very low.

It is noteworthy that the primers for amplification of the oac gene weredesigned based on the conserved regions in oac and oac_(1b) (which share88% identity) to guarantee both can be amplified. Although a serotype 5bstrain was not available, it can be predicted that the amplificationpattern of serotype 5b is 905 bp (gtrV) and 425 bp (gtrX) according tothe 0 antigen-modification characteristics of this serotype. As thereare not any typical strains of serotype 1c, a serotype F1 strain(untypable), 06HN081 (this strain can react with the 1c-specificmonoclonal antibody MASF1c), was used in the present invention to detectthe serotype 1c-specific gene gtrIC. As compared to typical 1c strains(which react exclusively with MASF1c), this F1 strain (untypable) canreact with both MASF1c and group 6 antisera. Accordingly, the genesgtrIC and oac that encode the antigen of MASF1c and the group 6 antigenwere both identified as positive. These results indicate that this F1(untypable) strain is converted from serotype 1b having its O-antigenmodified by GtrIC, and is therefore a new serotype (see Table 1). Thisnew serotype has the same serological reaction pattern as the strain ofserotype 7b reported in Forster et al., and thus the serotype of 06HN081should be 7b. It is to be noted that 1c is designated in Forster et al.as 7a (Foster, R. A., N. I. Carlin, M. Majcher, H. Tabor, L. K. Ng, andG. Widmalm. Structural elucidation of the O-antigen of the Shigellaflexneri provisional serotype 88-893: structural and serologicalsimilarities with S. flexneri provisional serotype Y394 (1c). CarbohydrRes 346:872-6). Hence, these two new serotypes can be identified by themultiplex PCR of the present invention. The present invention furtherprovides the use of the primers, the multiplex PCR method, and the kitfor identifying these two new serotypes.

Serotypes X and Xv have the same amplification pattern (wzx₁₋₅ andgtrX). Thus, they are classified as the same serotype Xv/X by themultiplex PCR method.

In order to evaluate the specificity of the primers, 50 non-Shigellaflexneri strains were tested in the examples, including other Shigellaserogroups and enteric pathogens. These bacteria all showed negativeafter the amplification (see FIG. 3), indicating the specificity of themethod of the present invention is 100%.

To determine whether the method of the present invention is applicableto all Shigella flexneri strains, and to assess the effectiveness of themethod of the present invention, 358 Shigella flexneri strains ofvarious serotypes were analyzed in the examples (see Table 2). Themultiplex PCR results of nearly all tested strains (except 8 strains)are consistent with the slide agglutination results, with a concordancerate of 97.8%.

TABLE 1 Serotype characteristics of S. flexneri reference strains byagglutination and multiplex PCR Serum Agglutination Reaction TypeAntigen Group Antigen MASF Multiplex PCR Strain No. Serotype □ □ □ IV V□ 3,4 6 7,8 1c wzx gtrI gtrIC gtrII oac gtrIV gtrV gtrX 2000019 1a + − −− − − + − − − + + − − − − − − 1997020 1b + − − − − − + + − − + + − − + −− − 06HN081^(#) — − − − − − − − + − + + + + − + − − − 301 2a − + − − −− + − − − + − − + − − − − NCTC4 2b − + − − − − − − + − + − − + − − − +03HL12 3a − − + − − − − + + − + − − − + − − + 2002110 3b − − + − − − − +− − + − − − + − − − NCTC9725 4a − − − + − − + − − − + − − − − + − −NCTC9726 4b − − − + − − − + − − + − − − + + − − 51247 5a − − − − + − + −− − + − − − − − + − 2003036 Y − − − − − − + − − − + − − − − − − −2001014 X − − − − − − − − + − + − − − − − − + 2002017 Xv − − − + − − −− + − + − − − − − − + 2000007 F6 − − − − − + − − − − − − − − − − − −^(#)Atypical F1 type, positive for both MASF1c and group 6 antiserum

TABLE 2 Correlation between the test results obtained from the multiplexPCR method and the slide agglutination method for 358 Shigella flexneristrains of various serotypes. Multiplex PCR No. of Target Gene serotypeSerotype Strains wzx₁₋₅ gtrI gtrIC gtrII oac gtrIV gtrV gtrXclassification F1a 25 25 25 0 0 0 0 0 0 1a (25) F1b 14 14 14 0 0 14 0 00 1b (14) F2a 55 55 0 0 55 0 0 0 0 2a (55) F2b 50 50 0 0 50 0 0 0 50 2b(50) F3a 10 10 0 0 0 10 0 0 10 3a (10) F3b 2 2 0 0 0 2 0 0 0 3b (2)  F4a5 5 0 0 0 0 5 0 0 4a (5)  F4b 5 5 0 0 0 5 5 0 0 4b (5)  F5a 4 4 0 0 0 10 4 0 5a (3), untypable (1) Y 36 36 0 0 5 0 0 0 0 Y (31), 2a (5) Xv 7878 0 0 0 0 0 0 78 X or Xv (78) X 69 69 0 0 2 0 0 0 69 X or Xv (67), 2b(2) F6 5 0 0 0 0 0 0 0 0 F6

TABLE 3  Primers Used in the Examples Length of Target AmplifiedSerotype Accession Gene Primer sequence (5′→3′) Fragments (bp)Specificity no. gtrI (F)CTGTTAGGTGATGATGGCTTAG 1122 (SEQ ID 1a, 1b, 1cAF139596 (SEQ ID No. 1) No. 19) (R)ATTGAACGCCTCCTTGCTATGC (SEQ ID No. 2)gtrII (F)ATTTATTGTTATTGGGGGTGGTTG 1268 (SEQ ID 2a, 2b AF021347(SEQ ID No. 3) No. 20) (R)ATTTGTTCTTTATTTGCTGGTT (SEQ ID No. 4) oac(F)CTGTTCGGCTTTGAAAGTGCTG 604 (SEQ ID 1b, 3a, 3b, AF547987(SEQ ID No. 5) No. 21) 4b (R)CGTAGGCGTACATAGCAAGCAAAGA (SEQ ID No. 6)gtrIV (F)ATGTTCCTCCTTCTTCCTTT 378 (SEQ ID 4a, 4b AF288197 (SEQ ID No. 7)No. 22) (R)TCCTGATGCTACCTTATCCA (SEQ ID No. 8) gtrV(F)AATACGATTCTCCTGGTGCTAAAC 905 (SEQ ID 5a, 5b U82619 (SEQ ID No. 9)No. 23) (R)TAGGGCATTGCTTGTATCTTTCAT (SEQ ID No. 10) gtrX(F)AATGCTGGATGGGATAATCACCTT 425 (SEQ ID 2b, 3a, 5b, L05001(SEQ ID No. 11) No. 24) X, Xv (R)GAGACGGCTTCTCCATGTTTTGCT(SEQ ID No. 12) wzx₁₋₅ (F) CACTTGTTGGGTATGCTGG 783 (SEQ ID 1-5, X, Xv,AE005674 (SEQ ID No. 13) No. 25) Y (R) CCGGCAAACAGATTAGAAA(SEQ ID No. 14) gtrIC (F) AGGGAATGGCATTAGGGATCGG 518 (SEQ ID 1c FJ905303(SEQ ID No. 15) No. 26) (R) GCTGCAAGTGGTTTTTGTTGGA (SEQ ID No. 16) wzx₆^(S) (F) TTAAGAGCGATCATTTC 739 (SEQ ID F6 EU294165 (SEQ ID No. 17)No. 27) (R) CCATCCAAGCGGACATT (SEQ ID No. 18) ^(S)The primer pair forwzx₆ is used to conform serotype F6.

The multiplex PCR method of the present invention may be employed toconduct biomolecular identification of Shigella flexneri serotypes inonly one reaction, and can easily and specifically identify the majorityof currently known serotypes (14 out of 15). In comparison with theconventional slide agglutination method (which requires as many as 10independent reactions), the multiplex PCR method of the presentinvention is time-saving, does not require expensive antisera, and isespecially suitable for high-throughput identification. Using thismethod, identification of 96 samples can be completed within 3.5 hoursand costs only 25% of that of the slide agglutination method.

The invention claimed is:
 1. A method for identifying Shigella flexneriserotypes, comprising performing amplification on a sample using a setof primers comprising the following primer pair nucleic acid sequences:SEQ ID NOs:1 and 2, SEQ ID NOs: 3 and 4, SEQ ID NOs: 5 and 6, SEQ IDNOs: 7 and 8, SEQ ID NOs: 9 and 10, SEQ ID NOs: 11 and 12, SEQ ID NOs:13 and 14, and SEQ ID NOs: 15 and
 16. 2. The method according to claim1, wherein the set of primers comprises the primer pair nucleic acidsequences of SEQ ID NOs: 17 and
 18. 3. The method according to claim 1,wherein the amplification is a polymerase chain reaction.
 4. The methodaccording to claim 3, wherein the polymerase chain reaction is amultiplex polymerase chain reaction.
 5. The method according to claim 4,further comprising performing singleplex PCR amplification on a sampleusing a pair of primers comprising the nucleic acid sequences of SEQ IDNOs: 17 and
 18. 6. The method according to claim 1, further comprisingperforming a qualitative analysis after the amplification.
 7. The methodaccording to claim 6, wherein the qualitative analysis comprisesvisualization of the amplified products through gel electrophoresis. 8.The method according to claim 1, further comprising conducting a slideagglutination reaction using anti-group IV antisera to furtherdifferentiate between serotypes Xv and X.
 9. The method according toclaim 1, wherein the sample is obtained from an isolated Shigellaflexneri strain.
 10. The method according to claim 1, wherein the sampleis an enriched or unenriched sample selected from the group consistingof: an excrement sample, an intestinal effusion sample, and a vomitsample.
 11. The method according to claim 1, wherein the sample is anenriched or unenriched sample selected from the group consisting of: awater sample, a soil sample, a food sample, and a cosmetic sample. 12.The method according to claim 1, wherein Shigella flexneri serotype 7aor 7b is identified by the amplification step.